MicroRNAs (miRNAs) are important post-transcriptional regulators of various biological pathways. In this study, we focused on the role of miRNAs during mitochondrial biogenesis in skeletal muscle. The expression of miR-494 was markedly decreased in murine myoblast C2C12 cells during myogenic differentiation, accompanied by an increase in mitochondrial DNA. Furthermore, the expression of predicted target genes for miR-494, including mitochondrial transcriptional factor A (mtTFA) and Forkhead box j3 (Foxj3), was post-transcriptionally increased during myogenic differentiation. Knockdown of miR-494 resulted in increased mitochondrial content and upregulation of mtTFA and Foxj3 at the protein level. A 3'UTR reporter assay revealed that miR-494 knockdown directly upregulated the luciferase activity of mtTFA and Foxj3. All of these observations were reversed by overexpression of miR-494. Furthermore, the miR-494 content significantly decreased after endurance exercise in C57BL/6J mice, accompanied by an increase in expression of mtTFA and Foxj3 proteins. These results suggest that miR-494 regulates mitochondrial biogenesis by downregulating mtTFA and Foxj3 during myocyte differentiation and skeletal muscle adaptation to physical exercise.